1. Field of the Invention
This invention relates to electrophoresis devices, and more particularly, to a novel gel displacement type comb for such devices.
2. Description of Related Art
Electrophoresis is a technique that is currently used in the separation of proteins, lipoproteins, DNA, and RNA. This technique involves the separation of charged macromolecules due to their differential mobilities through a porous gel under an applied electric field. Commonly used gels are polyacrylamide and agarose.
Usually, electrophoresis separation can be based on differences in molecular weight, and is a particularly powerful tool for separation or fractionation of macromolecule mixtures in which the molecular weights of the molecules are from about 10,000 to about 10 million or more daltons.
Gel electrophoresis devices may be classified into two general types: vertical and horizontal. Both types have been well documented in the literature.
For the application of samples for gel electrophoresis, a sample well-forming device (commonly referred to as a "comb") is used. As illustrated in FIG. 1, a standard comb 10 is generally of a rectangular shape, from 2 to 5 cm in width and 5 to 20 cm in length, and flattened (from 0.2 mm to 2 or 3 mm thick), having along one long side a plurality of rectangular shaped protrusions (or "teeth") 12, each being of about 2.5 to 10 mm or more in width, separated by generally narrower (i.e., 1.6-3 mm) rectangular indentions 13 (or spaces). The standard comb is made of a plastic material. Delrin (or acetal), lexan, and acrylic, among others, have been used as the plastic material. In one common form of prior art applicable to both vertical and horizontal gel formats, such combs are inserted into the still liquid gel medium, which flows into the spaces between the teeth and then solidifies. When the comb is removed, a void is left in the gel matrix in the place where the teeth had excluded the liquid gel medium. This void is referred to as the "sample well."
Vertical and horizontal gels differ in the relationship of the comb, the sample wells, and the direction with respect to these that the sample travels during electrophoresis. Vertical gels are formed between two rectangular glass plates separated by a spacer of a given thickness (0.2 mm to 3 mm or slightly more, and nominally equal to the thickness of the comb) which commonly has three closed (and sealed) sides and one open side. The gel material is poured into the open side, filling the space between the plates to within a few mm of the edge of the open side. The comb is then introduced between the plates at the open side and pushed down into the liquid gel until its teeth become at least partially, or more commonly, completely submerged in the liquid gel medium.
Upon removal of the comb from the solidified gel medium, rectangular shaped voids (wells) are left at the edge of the gel. Such voids are delineated at the front and rear by the front and rear glass plates respectively, and at the sides and bottom by the solidified gel medium itself. "Fingers" of solidified gel medium (also referred to as "well dividers") formed from liquid gel that flowed into spaces between the comb teeth serve to separate the sample wells from each other. When electric current is applied, the sample migrates parallel to the plane of the comb (now removed), toward the floor of the vertical well, and from there through the length of the gel medium, toward the cathode.
In horizontal gels, the liquid gel (most often agarose) is poured to a given thickness (usually 4 to 5 mm) onto a horizontal, flat, rectangular plate, bounded on all four sides by walls which serve to contain the gel medium (i.e., a "tray"). The comb is orientated perpendicular to the plane of the horizontal gel, near one side of the gel (which will become the anodic side during electrophoresis), and held firmly in place with the bottom of the teeth suspended approximately 1 mm above the base of the tray, usually supported by the side walls of the tray using a bridging device. The liquid gel medium flows around the teeth and into the spaces between the teeth and solidifies. Upon removal of the comb, voids are left in the gel as sample wells, which are delineated entirely by gel medium--on both broad faces, the floor and the two sides. Wells are separated from one another by the solidified gel medium which occupied the spaces between the teeth of the comb. During electrophoresis, samples migrate perpendicular to the plane of the comb, exiting the broad face of the well on the cathodic side and migrating through the gel medium towards the cathode.
A significant shortcoming of the prior art relative to the introduction of sample wells into vertical and horizontal gel formats is the limited proximity of adjacent wells that can be achieved with conventional combs. Maximum proximity is desirable since it affords a more accurate comparison of the mobilities of samples in neighboring lanes, and allows more sample wells of any particular width to be accommodated within a gel of given overall width. This problem arises with conventional combs because to assure the structural integrity of the well, a given minimum thickness of gel medium must be provided to divide individual wells. This is particularly true given that both polyacrylamide and (particularly) agarose gel matrices posses rather limited structural strength. The problem is further exacerbated by the stress forces placed upon the fragile gel dividers through the necessary action of removing the comb from the solidified gel. This stress derives from the frictional drag of the comb in contact with the gel and the partial vacuum generated upon removal of the comb, since adhesion of the gel to the comb hinders pressure equalization by blocking the inflow of air into the well.
In the case of vertical polyacrylamide gels, this shortcoming has been addressed and eliminated by the use of a comb which functions using a substantially different principle from that of the conventional gel displacement combs described above. This comb, called the "sharkstooth" comb (devices of U.S. Pat. Nos. 4,883,577 and 5,164,065) consists of sharply pointed teeth which are pressed firmly against the already solidified edge of the gel medium (exclusively polyacrylamide) sandwiched between two glass plates. The spaces between the teeth are not allowed to pushed completely between the glass plates, but are rather allowed to extend above the plate. This allows access for sample loading through the open side of the sandwiched plates, to the spaces above the gel surface, which corresponds to the spaces between the comb's teeth. Such spaces are delineated on each side by the edges of adjacent teeth and on the bottom by the surface edge of the gel medium. Thus, in this case, it is the spaces between the teeth, rather than gel medium displaced by the body of the teeth, which form the sample wells.
Unlike gel displacement combs, the sharkstooth comb is not withdrawn since this would negate its well-forming function, but rather stays in place during sample loading and at least until all sample has entered the gel matrix. Since the sharksteeth narrow to a point (where the points of the teeth make contact with the gel surface), adjacent lanes are formed in close proximity (essentially touching).
Importantly, the sharkstooth principle cannot be used with horizontal gels since horizontal gel formats of necessity require gel displacement type wells to be formed. Until now, no attempt has been made to bring the same advantages of close proximity wells to the horizontal gel format using a gel displacement comb. The present invention addresses this need by providing novel geometry of the teeth of a gel displacement type comb for horizontal gel electrophoresis.